Bioreactors, also referred to as fermentors, are a part of biotechnological apparatuses. They have a closed reaction chamber, in which eukaryotic or prokaryotic cells are cultivated under conditions that are as optimal, defined and controlled as possible. Conversions of substances, mostly automated and controlled by process engineering, are researched, optimised and performed using the boundary conditions necessary for the organism and in the presence of the primary and secondary substances required for the process.
Biotechnological cultivation is carried out under conditions that are optimised for the organism and for the conversions to be achieved. For a large proportion of the cell types used, this process takes place at 37° C., at which temperature dry air can absorb around 40 grams of water per kilogram of air. This means that water is continuously withdrawn from the system when the culture is gassed with the gas composition required for the microorganisms. In order to counteract this effect, the exhaust gas stream is guided through the exhaust gas conduit provided with the temperature control device which cools the surface of the exhaust gas conduit, which then cools the gas stream, for example to a temperature between about 4° C. and about 10° C. By this means, the exhaust gas stream is cooled down significantly. The temperature on the surface of the exhaust gas conduit falls below the dew point. As a result of this measure, the water being removed in the exhaust gas stream condenses on the walls of the exhaust gas cooler and runs back into the culture medium.
When using such bioreactors in the field of cell culture technology, cultivation times ranging from a number of days to a few weeks are not atypical. The gassing rates are also significantly lower than in microbiological applications. For this reason, it may also be necessary to heat the exhaust gas stream in order to prevent clogging of the sterile filter connected downstream from the exhaust gas conduits.
Controlling the temperature by means of the temperature control device is typically done using a temperature control fluid. This fluid is conditioned in a separate unit. Mains water may also be used as an alternative if its temperature is sufficiently controlled.
When using coolants and separate units, complex connection technology is needed. The different temperatures of the surroundings and the connector units result in many temperature divergences, which is why strong formation of condensate on supply and discharge pipelines can be observed.
In typical biotechnological methods carried out on “benchtop scale” (laboratory scale), glass reaction vessels are frequently used. This allows an autoclavable bioreactor to be designed in which the glass reactor vessel can be steam sterilised in one piece in an autoclave. In this context, it is then necessary that all connections to the reaction vessel be disconnected from the control units before autoclaving. Connections are typically in the form of hose connectors such as clamp screw connections, push-in connectors, crimp connections and the like. Such an autoclavable design of the bioreactor thus requires that functional components of the biotechnological apparatus which connect to the bioreactor itself are mounted on the reactor vessel as efficiently as possible and can be dismantled again when autoclaving is due.
Another bioreactor design takes the form of single-use bioreactors, in which the reactor vessel, for example, is used in one cultivation process only, whereas functional elements assigned to the reactor vessel, such as stirrer drives, or the temperature control unit for exhaust gas removal, can be reused. In this connection also, it is necessary that functional components of the biotechnological apparatus which are coupled to the bioreactor be mounted on the reactor vessel and dismantled again after use as efficiently as possible.
Bioreactors, which are also frequently referred to as fermenters, enclose a reaction chamber in which biological or biotechnological processes can be carried out on a laboratory scale. Such processes include, for example, the cultivation of cells, microorganisms or small plants under defined, preferably optimized, controlled and reproducible conditions. Bioreactors mostly have a plurality of connectors via which the primary and secondary substances, as well as various instruments, such as sensors, can be introduced to the reaction chamber, or to which fluid conduits, for example, can be connected. Fluids, in particular gases, can be supplied to or removed from the reaction chamber through such fluid conduits, in particular gas conduits. Depending on the direction of fluid flow, fluid conduits can be referred to as feed conduits or discharge conduits. Gas conduits, for example, can be referred to as gas feed or gassing lines or conduits or as gas exhaust or discharge conduits, depending on the direction of the gas stream.
Bioreactors are preferably used in bioreactor systems, preferably in parallel bioreactor systems, not only in the field of cell cultivation but also in microbiological applications. Parallel bioreactor systems are described in DE 10 2011 054 363.5 and DE 10 2011 054 365.1, for example. In such a bioreactor system, a plurality of bioreactors can be operated in parallel and controlled with higher precision. High-throughput experiments that are well reproducible and scalable can be carried out in the individual bioreactors, even with small operating volumes.
In the cell culture field, such parallel bioreactor systems are used, for example, for test series for process optimization based on statistical planning methods (design of experiments DoE), for process development and in research and development, for example to cultivate different cell lines such as Chinese hamster ovary (CHO), hybridoma or NSO cell lines. In the context of the present application, the expression “cell culture” is specifically understood to mean the cultivation of animal or plant cells in a nutrient medium outside the organism.
In the field of microbiology, parallel bioreactor systems are likewise used for test series for process optimization based on statistical planning methods (design of experiments DoE), for process engineering and in research and development, for example to cultivate various microorganisms, in particular bacteria or fungi, such as yeast.
Bioreactors used in laboratories are often made of glass and/or metal, in particular of stainless steel, as the bioreactors must be sterilized between different uses, preferably by steam sterilization in autoclaves. Sterilizing and cleaning reusable bioreactors is a complex process. The sterilization and cleaning process can be subject to validation, and needs to be precisely documented for each individual bioreactor. Residues in a bioreactor which has not been fully sterilized can falsify the results of a subsequent process, or render them useless, and may cause disruption of the subsequent process. Furthermore, the sterilization process may also expose individual components or materials in bioreactors to stress and strain, and in some cases can damage them.
Single-use bioreactors provide an alternative to reusable bioreactors and are used to carry out just one biological or biotechnological process before being disposed of. By providing a new single-use bioreactor for each process, and one that is preferably sterilized during the production process, it is possible to reduce the risk of (cross-) contamination, while simultaneously obviating the need to perform and document the impeccable cleaning and sterilization of a previously used bioreactor. Single-use bioreactors are often designed as flexible containers, for example as bags, or as containers having walls that are flexible in sections thereof at least. Examples of such bioreactors are described in US 2011/0003374 A1, US2011/0058447A1, DE 20 2007 005 868U1, US 2011/0058448A1, US2011/0207218A1, WO 2008/088379A2, US 2012/0003733 A1, WO2011/079180A1, US2007/0253288A1, US 2009/0275121A1 and US 2010/0028990A1. Dimensionally stable single-use bioreactors are known from EP 2 251 407 A1 and from US 2009/0311776 A1, for example.
A sterile single-use bioreactor generally includes a fluid conduit attached thereto, which is likewise sterilized and provided for single use, in particular one or more gas conduits. Such a single-use fluid conduit may be embodied as a rigid pipe or as a flexible tube. A sterile filter which in most cases is likewise sterilized and provided for a single use is usually provided on a single-use fluid conduit, preferably at the end which is not attached to the bioreactor. In a gas conduit in particular, a sterile filter is used to filter gas flowing into a bioreactor through a gassing conduit, or gas flowing out of a bioreactor via an exhaust gas conduit.
When using bioreactors in biological or biotechnological processes, for example to cultivate various microorganisms, in particular bacteria or fungi, such as yeast, or for cultivating various cell lines such as Chinese hamster ovary (CHO), hybridoma or NSO cell lines, it is important that the sterility of the single-use bioreactor and its components is not compromised. In the context of the present application, the expression “cell culture” is specifically understood to mean the cultivation of animal or plant cells in a nutrient medium outside the organism.
Such biological and biotechnological processes are generally carried out under sterile conditions in aqueous media, also referred to as culture broths, in a reaction chamber of a bioreactor. Gaseous fluid, for example mixtures of nitrogen, oxygen, carbon dioxide, etc. are fed to the system during at least a certain period of cultivation in order to keep the biological processes running, and/or gaseous fluids such as methane, carbon dioxide, etc. are removed from the system that may also contain other components produced by biological processes. The sterile conditions in the reaction chamber of a bioreactor and hence also of the biological system, both when supplying fluids and also when removing fluids, are generally maintained by sterile filters inserted into the fluid stream. The gaseous fluid which is removed is typically saturated with water vapor. The temperature of the aqueous culture broth is mostly higher than the temperature of the system's environment, for example in a laboratory. In such cases, the removed gaseous fluid is cooled at least slightly when discharged from the reaction chamber of the bioreactor into the exhaust gas tube. This causes part of the water vapor in the gas to condense. It is desirable to return the condensate to the reaction chamber, because otherwise the concentration of media components in the culture broth could increase in the course of the process, which can lead, for example, to an undesirable increase in osmolality. Another disadvantage is that the sterile filter may be blocked by the formation of condensate, as a result of which the gaseous fluid can no longer be removed, thus causing the exhaust gas stream to be blocked. This can lead to the gas supply into the reaction chamber being blocked as well, thus disrupting or terminating the cultivation process.
Cooling devices are therefore deployed in the exhaust gas conduits in reusable bioreactors, as shown, for example, in US 2012/0103579 A1, US 2010/0170400 A1 and US 2011/0076759 A1. An exhaust gas cooling device for single-use bioreactors is proposed in WO 2011/041508 A1, for example.
Due to the need to preserve sterility, in particular, the use of systems for cooling exhaust gas has not yet become established practice in single-use bioreactors, however, which can lead to the aforementioned disadvantages of water extraction and filter blocking. In some existing solutions, the attempt is made to prevent blocking of a sterile filter by lowering the relative moisture in the exhaust gas stream to such an extent, by heating the exhaust gas in the region of the exhaust gas tube, that the exhaust gas can pass through the sterile filter without condensate being produced. This does not do away with the disadvantage that no condensate is recovered from the exhaust gas that could be returned to the culture solution. Moreover, blocking of the sterile filter can still occur when these solutions are applied. In order to increase process reliability, complex single-use sterile filters such as depth filters or capsule filters are often used, therefore, instead of membrane filters, which are inexpensive but have a tendency to clog. However, this results in higher costs for single-use bioreactors.
It is also tried to avoid blocking of the sterile filter by means of heating the sterile filter itself, by increasing the temperature in the sterile filter to avoid or reduce the forming of condensate. This also, inter alia, has the disadvantage that no condensate is recovered from the exhaust gas that could be returned to the culture solution, and also this approach cannot reliably enough prevent a blocking of the sterile filter.
A biotechnological device with a temperature control device is known from the patent application DE 10 2011 054 364.3. Although the biotechnological device described therein and the temperature control device likewise described therein provide advantages for bioreactors which can be autoclaved, it is nonetheless desirable to further simplify and improve temperature control of exhaust gases for sterile, single-use bioreactors, particularly with regard to process reliability and energy efficiency. The sterility of a single-use bioreactor and of a sterile disposable exhaust gas tube attached thereto should not be adversely affected by providing a device for controlling the temperature of the exhaust gas.
Another disadvantage of prior art solutions is that the temperature in the reaction chamber can be influenced or changed by feeding fluids or gases, for example cooled fluids, into the reaction chamber, which can have negative effects on temperature-sensitive processes especially, and may make it more complex or expensive to control the temperature of the culture broth. Also, the temperature control can be negatively influenced by feeding fluids or gases, for example cooled fluids, into the reaction chamber, particularly by cold gas impulses. Gas supply or gassing conduits may likewise be fitted with filters that may become blocked as a result of condensate forming. When feeding moist gas or gas mixtures, for example when feeding the exhaust gas from an upstream process as a feed gas to a downstream bioreactor, it may be desirable to filter out gas components by means of a filter, for which blocking of the filter is likewise disadvantageous.